This invention relates to the use of part of tetanus toxin for delivering a composition to the central nervous system of a human or animal. This invention also relates to a hybrid fragment of tetanus toxin, a polynucleotide that hybridizes with natural tetanus toxin, and a composition containing the tetanus toxin fragment as an active molecule. Further, this invention relates to a vector comprising a promoter and a nucleic acid sequence encoding the tetanus toxin fragment. In addition, this invention relates to methods of using the tetanus toxin fragment.
Tetanus toxin is produced by Clostridium tetani as an inactive, single, polypeptide chain of 150 kD composed of three 50 kD domains connected by protease-sensitive loops. The toxin is activated upon selective proteolytic cleavage, which generates two disulfide-linked chains: L (light, 50 kD) and H (heavy, 100 kD) [Montecucco C. and Schiavo G. Q. Rev. Biophys., (1995), 28: 423-472].
Evidence for the retrograde axonal transport of tetanus toxin to central nervous system (CNS) has been described by Erdmann et al. [Naunyn Schmiedebergs Arch Phamacol., (1975), 290:357-373], Price et al. [Science, (1975), 188:945-94], and Stoeckel et al. [Brain Res., (1975), 99:1-16]. In each of these studies, radiolabeled toxin was found inside membrane bound vesicles. Another property was the transynaptic movement of tetanus toxin that was demonstrated first by autoradiographic localization of 125I-labeled tetanus toxin in spinal cord interneurons after injection into a muscle [Schwab and Thoenen, Brain Res., (1976), 105:218-227].
The structure of this tetanus toxin has been elucidated by Helting et al. [J. Biol. Chem., (1977), 252:187-193]. Papain cleaves the tetanus toxin in two fragments:                the C terminal part of the heavy chain, 451 amino acids, also called fragment C; and        the other part contained the complementary portion called fragment B linked to the light chain (fragment A) via a disulfide bond.        
European Patent No. EP 0 030 496 B1 showed the retrograde transport of a fragment B-IIb to the CNS and was detected after injection in the median muscle of the eye in primary and second order neurons. This fragment may consist of “isofragments” obtained by clostridial proteolysis. Later, this fragment B-IIb was demonstrated to be identical to fragment C obtained by papain digestion by Eisel et al. [EMBO J., 1986, 5:2495-2502].
This EP patent also demonstrated the retrograde transport of a conjugate consisting of a Ibc tetanus toxin fragment coupled by a disulfide bond to B-IIb from axonal endings within the muscle to the motoneuronal perikarya and pericellular spaces. (The Ibc fragment corresponds to the other part obtained by papain digestion as described above by Helting et al.). There is no evidence that this conjugate was found in second order neurons. The authors indicated that a conjugate consisting of the fragment B-IIb coupled by a disulfide bond to a therapeutic agent was capable of specific fixation to gangliosides and synaptic membranes. No result showed any retrograde axonal transport or a transynaptic transport for such conjugate.
Another European Patent, No. EP 0 057 140 B1, showed equally the retrograde transport of a fragment IIc to the CNS. As in the European Patent No. EP 0 030 496 B1, the authors indicated that a conjugate consisting of the fragment IIc and a therapeutic agent was capable of specific fixation, but no result illustrated such allegation. This fragment IIc corresponds to the now called fragment C obtained by papain digestion.
Francis et al. [J. Biol. Chem., (1995), 270(25):15434-15442] led an in vitro study showing the internalization by neurons of hybrid between SOD-1 (Cu Zn superoxide dismutase) and a recombinant C tetanus toxin fragment by genetic recombination. This recombinant C tetanus toxin fragment was obtained from Halpern group. (See ref. 11).
Moreover, Kuypers H. G. J. M and Ugolini G. [TINS, (1990), 13(2):71-75] indicated in their publication concerning viruses as transneuronal tracers that, despite the fact that tetanus toxin fragment binds to specific receptors on neuronal membranes, transneuronal labeling is relatively weak and can be detected only in some of the synaptically connected neurons.
Notwithstanding these advances in the art, there still exists a need for methods for delivering compositions into the human or animal central nervous system. There also exists a need in the art for biological agents that can achieve this result.
Additionally, activity-dependent modification of neuronal connectivity and synaptic plasticity play an important role in the development and function of the nervous system. Recently, much effort has been dedicated to following such modifications by the engineering of new optically detectable genetic tools. For example, fused to a reporter gene such as LacZ or GFP (Green Fluorescent Protein), the atoxic C-terminal fragment of tetanus toxin (or TTC fragment) can traffic retrogradely and transsynaptically inside a restricted neural network either after direct injection of the hybrid protein (Coen et al., 1997), or when expressed as a transgene in mice (Maskos et al., 2002). The dynamics of βgal-TTC clustering at the neuromuscular junction (NMJ) is strongly dependent on a presynaptic neuronal activity and probably involves fast endocytic pathways (Miana-Mena et al., 2002). Neuronal activity may induce this clustering and internalization at the NMJ by enhancing the secretion and/or action of various molecules at the synapse.
Over the past decade, various data indicate that neurotrophins, a family of structurally and functionally related proteins, including NGF (Nerve Growth Factor); BDNF (Brain Derived Neurotrophic Factor); Neurotrophin 3 (NT-3) and Neurotrophin 4 (NT-4), not only promote neuronal survival and morphological differentiation, but also can acutely modify synaptic transmission and connectivity in central synapses, thus providing a connection between neuronal activity and synaptic plasticity (McAllister et al., 1999; Poo, 2001; Tao and Poo, 2001). The role of these factors in neurotransmission between motoneurons and skeletal muscle cells has been studied using Xenopus nerve-muscle co-culture studies, whereby the treatment of these cultures with exogenous BDNF, NT-3 or NT4 leads to an increase of synaptic transmission by enhancing neurotransmitter secretion (Lohof et al., 1993; Stoop and Poo, 1996; Wang and Poo, 1997). Moreover, the muscular expression of NT-3 and NT-4 (Funakoshi et al., 1995; Xie et al., 1997), as well as NT-4 secretion (Wang and Poo, 1997) are regulated by electrical activity. This family of proteins thus provides a molecular link between electrical neuronal activity and synaptic changes.
The cellular actions of neurotrophins are mediated by two types of receptors: the p75NTR receptor, mainly expressed during early neuronal development, and a Trk tyrosine kinase receptor (Bothwell, 1995). The interaction of neurotrophins with Trk receptors is specific. TrkB and TrkC, are activated by BDNF/NT-4 and NT-3, respectively, and are expressed by motor neurons. TrkA, which is expressed by sensory neurons, is activated by NGF. Recently, evidence for a co-trafficking between TTC and the neurotrophin receptor p75NTR has been reported in cultured motoneurons (Lalli and Schiavo, 2002), as well as the activation by tetanus toxin and the TTC fragment of intracellular pathways involving Trk receptors in cultured cortical neurons (Gil et al., 2003).
Notwithstanding the knowledge in the art, there still exists a need for understanding the influences of neurotrophins and other neurotrophic factors on TTC traffic at the NMJ in vivo and for developing methods of using these neurotrophins and neurotrophic factors, and agonists or antagonists thereof, to modulate the neuronal transport of a tetanus toxin or a fusion protein comprising a fragment C of the tetanus toxin.
To date, active synapses have often been characterized by their electrical properties using electrophysiological recordings of patched neurons. Thus, depending on the electrical response of cells to different solutions, it has been possible to study indirectly active synapses and the activity of membrane receptors specific of presynaptic or postsynaptic endings. However, direct visualization of active synapses in complex neuronal networks has been elusive to date because of the lack of biological markers being specifically addressed and/or accumulated in these neuronal structures.
The recent development of styryl dyes has revolutionized the way in which functional synapses can be studied, inasmuch as they are excellent reagents both for identifying actively firing neurons and for investigating the mechanisms of activity-dependent vesicle cycling in a broad list of species. Thus, in a neuron that is actively releasing neurotransmitters, these dyes become internalized within the recycled synaptic vesicles and the nerve terminals become brightly stained (Cochilla, 1999). Nevertheless, the non-proteic nature of these molecules and the transient labeling that they provide represent difficult caveats when trying to study long-term synaptic activity and remodeling. Moreover, staining of more physiological relevant networks such as those presented in brain slices or intact animals with styryl dyes has been proven to be arduous, requiring the reduction of nonspecific background fluorescence while preserving the specific fluorescent signal by adding sulforhodamine or other fluorescent quenching reagents (Pyle, 1999).